Fottea, Olomouc, 12(2): 231–253, 2012 231 Genotypic diversity of Dictyosphaerium–morphospecies (Chlorellaceae, Trebouxiophyceae) in African inland waters, including the description of four new genera Lothar KRIENITZ1*, Christina BOCK1,2, Kiplagat KOTUT3 & Thomas PRÖSCHOLD4,5 1Leibniz–Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhütte 2, D–16775 Stechlin–Neuglobsow, Germany; *Corresponding author e–mail: krie@igb–berlin.de 2University of Essen, Faculty of Biology, D–45141 Essen, Germany 3Plant and Microbial Sciences Department of Kenyatta University Nairobi, PO Box 43844 GPO 00100 Nairobi, Kenya 4University of Vienna, Department of Limnology, Althanstr. 14, A–1090 Vienna, Austria 5University of Rostock, Department of Applied Ecology, Albert–Einstein–Str. 3, D–18059 Rostock, Germany Abstract: Trebouxiophytes of the Dictyosphaerium–morphotype from inland waters of Africa were studied using a polyphasic approach of SSU and ITS rDNA phylogeny, secondary structure of the ITS and observations made with a light microscope. Although the morphological criteria for differentiating species and genera are scarce, the genetic diversity of these algae was very high. Based on our genetic analysis findings, we described four new genera containing five new species: Compactochlorella dohrmannii, Compactochlorella kochii, Kalenjinia gelatinosa, Marasphaerium gattermannii and Masaia oloidia. Diversity and distribution of Chlorella–related colonial chlorophytes in the tropical and temperate zones were compared and discussed. Key words: Inland waters of Africa, Chlorella, Compactochlorella nov. gen., Dictyosphaerium, diversity, geographic distribution, green algae, Kalenjinia nov. gen., Marasphaerium nov. gen., Masaia nov. gen. Introduction and recommended studies using a polyphasic approach combining morphological and molecular Coccoid green algae are among the most diverse phylogenetic methods. microphytes. In their famous handbook, KOMÁREK Studies about algal biodiversity in Africa & FOTT (1983) compiled more than 1200 taxa. have been hampered by several circumstances. For However, these taxa are defined by morphology. more than 100 years, algae in phytoplankton have To discover the real genotypic diversity of been investigated. However, a general limitation of coccoid green algae only few reports with limited this classical research, especially in the early years progress have been made. For example, POTTER is that only fixed samples were taken by scientific et al. (1997) came up with a thesis of convergent travellers and later studied in various laboratories. evolution of morphology that masks the extensive These findings can be brought into agreement biodiversity among coccoid picoplankton. FAWLEY only with difficulties with the today’s views over et al. (2004) studied the molecular diversity of the systematics of algae. The second half of the coccoid and monadoid green algae in 20 lakes of 20th century saw an increase in the documentation North America and found 93 distinct SSU rRNA of detailed morphological characteristics leading genotypes, 89 of those were new to science. to the generation of data useful for comparative FAWLEY et al. (2005) evaluated the morphospecies studies. The most usable findings were published concept in Selenastraceae and concluded that on lakes of the southern and eastern Africa numerous cryptic species are hidden by one and (HUBER–PESTALOZZI 1929; TALLING 1987; COCQUYT the same morphotype. PRÖSCHOLD & LELIAERT et al. 1993). However, the majority of systematic (2007), COESEL & KRIENITZ (2008) and RINDI et al. considerations on material from tropical countries (2010) discussed the state of the art in diversity are based on identification keys established for and taxonomic conceptions in green algae temperate zone taxa. Furthermore, investigations 232 KRIENITZ et al.: Genotypic diversity of Dictyosphaerium on living and cultured algal material including water chemistry or the strong interaction with sequence analyses are largely missing. Until now, consumer and decomposer populations in the only few cultured strains of coccoid green algae waters studied. The question that remains very from tropical Africa have been investigated by contentious is the amount of morphological modern systematic approaches (LUO et al. 2006, differences that are necessary to reflect the 2010; BOCK et al. 2010, 2011a; KRIENITZ & BOCK genotypic diversity of these Dictyosphaerium– 2011; KRIENITZ et al. 2010, 2011). like green algae. Molecular phylogenetic It is widely accepted that microbial diversity studies have shown that the Dictyosphaerium– differs fundamentally from biodiversity of larger morphotype evolved independently in different animals and plants (NORTON et al. 1996). Some clades of the Chlorophyta (BOCK et al. 2010, workers, including FENCHEL & FINLAY (2004), 2011b; KRIENITZ et al. 2010, 2011). Out of a have taken up the hypothesis of BAAS–BECKING collection of 27 chlorellacean strains isolated (1934) based on a metaphor by BEIJERINCK, from different inland waters of Africa, 24 strains which suggests that free–living microbes have a belonged to the Dictyosphaerium–morphotype. cosmopolitan distribution and that most protistan Based on these strains this study will address the organisms (microalgae and protozoa), smaller questions (i) how diverse are these coccoid green than one millimeter in size, have a worldwide algae in terms of morphology and phylogeny, distribution wherever their required habitats are and (ii) are the tropical Dictyosphaerium–like realised (“everything is everywhere, but, the algae genotypically identical with those from environment selects” – see DE WIT & BOUVIER the temperate climatic region? The outcome of 2006). This is a result of their ubiquitous dispersal this study is the description of four new genera driven by huge population sizes, and consequently including five new species. a low probability of local extinction. However, this hypothesis is only based on the phenotypic (“morphospecies”) approach and depends on Material and Methods clear identification of the microorganisms. For microalgae, the hypothesis is controversial and In this study 67 strains belonging to the class remains the subject of ongoing discussions Trebouxiophyceae were used (Table 1). Out of 28 (COLEMAN 2002; FINLAY & FENCHEL 2002; FOISSNER African strains, 26 strains were isolated by one 2006). Further studies, including more isolates of the authors from eight inland waters of Kenya, from different regions of the world, are needed and one or two waters of Angola, Tanzania, to prove or reject the hypothesis on the universal Tunisia, Uganda, and Zambia, respectively. These dispersal of microalgae. habitats were of different characteristics. Beside One of the most common morphotypes of the large Lake Victoria, smaller lakes of the Rift coccoid green algae in the phytoplankton of inland Valley, the lakes Baringo, Naivasha and Oloidien waters is represented by members of the genus were chosen as sampling area. Other strains were Dictyosphaerium. The genus is characterized isolated from ephemeral pools in the Ngorongoro by green spherical cells interconnected by Crater in Tanzania, Nakuru National Park in mucilaginous strands in colonies surrounded by Kenya and from a park pond in Nairobi. We also thick gelatinous envelopes (Fig. 1). Although this isolated strains from sewage oxidation ponds, morphotype has a world–wide distribution, it is such as the final sewage pond of the Nakuru town difficult to compare findings from different climatic sewage plant, and from a sewage pond on the regions. On the base of their morphology, about 11 Djerba island in Tunisia. Additional strains were species are known (KOMÁREK & PERMAN 1978). The collected in rivers or water channels such as the type species Dictyosphaerium ehrenbergianum Mara river in the Masai Mara National Reserve NÄGELI was found in European waters, and closely in Kenya, from the Kunene and Okawango in related morphotypes were also identified as very Angola, and from the Kazinga channel in Uganda, common in tropical inland waters. Microscopic which is connected to lakes Albert and George. studies have revealed that in comparison to the We isolated single Chlorella–like cells or phytoplankton from other continents, samples colonies of the Dictyosphaerium–morphotype from Africa appear to be especially rich in by glass–capillaries from the field samples and specimens with thick mucilaginous envelopes. transferred them to a liquid medium. All strains This unusual phenomenon may be a response to were maintained at the strain collection of the Fottea, Olomouc, 12(2): 231–253, 2012 233 IGB (Leibniz–Institute of Freshwater Ecology For the first phylogenetic tree, the SSU was and Inland Fisheries). The strains were grown manually aligned on the basis of the predicted in a modified BOURRELLY medium (HEGEWALD et secondary structure model for Micractinium al. 1994; KRIENITZ & WIRTH 2006) on agar at 15 pusillum (LUO et al. 2006). Stems and loop regions °C or in suspensions at room temperature under of the 18S of the strains were aligned to each other a 14 h : 10 h light–dark regime. New strains were respectively. The ITS regions were more difficult deposited at the Culture Collection of Algae and to align due to a high degree of divergence between Protozoa (CCAP, Oban, UK). The algae were the sequences. Within this regions, we aligned investigated using a Nikon Eclipse E600 light them strictly according to their predicted secondary microscope with differential interference